Recombinant equine herpesvirus-1 vaccine containing mutated glycoprotein C and uses thereof

ABSTRACT

The present invention provides compositions or vaccines that contain a recombinant EHV-1 that elicit an immune response in animals against equine herpesvirus, including compositions comprising the recombinant EHV-1, methods of vaccination against equine herpesvirus, and kits for use with such methods and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/840,935 filed on Mar. 15, 2013, which claims benefit of U.S.provisional application Ser. No. 61/613,151 filed Mar. 20, 2012.

FIELD OF THE INVENTION

The present invention relates to compositions or vaccines for combatingequine herpesvirus infections in animals. Specifically, the presentinvention provides compositions or vaccines that contain a recombinantEHV-1 that elicit an immune response in animals against equineherpesvirus, including compositions comprising said recombinant EHV-1,methods of vaccination against equine herpesvirus, and kits for use withsuch methods and compositions.

BACKGROUND OF THE INVENTION

Equine Herpesvirus type 1 (EHV-1) is one of the most important andprevalent pathogens of equine populations worldwide (Ma et al., J. ofGeneral Virology 91, 1817-1822, 2010). Together with its close relativesvaricella-zoster virus, bovine Herpesvirus type 1, pseudorabies virusand EHV-4, EHV-1 forms the genus Varicellovirus in the subfamilyAlphaherpesvirinae of the family Herpesviridae of the orderHerpesvirales (Davison et al., The order Herpesvirales. Arch Virol 154,171-177, 2009). Diseases caused by EHV-1 range from mildrhinopneumonitis and abortion in pregnant mares to neurological diseasethat is frequently lethal in affected horses (Allen et al., Prog VetMicrobiol Immunol 2, 78-144, 1986; Carroll et al., Aust Vet J 62,345-346, 1985; Crabb et al., Adv Virus Res 45, 153-190, 1995). Thepathogenesis of EHV infection is very complex. Natural infection occursthrough inhalation or ingestion of the infectious virus. Within a fewdays of the virus can be found in leucocytes, where it is protected fromrecognition and attacks by the immune system. The virus disseminates viacell-associated viremia to secondary sites of replication (Allen et al.,Proceedings 8^(th) Equine Infectious Disease Conference, Dubai 23-26, pp129-146, 1998).

EHV-1 harbors a 150 kb double-stranded DNA genome that is highlyconserved among strains. A neuropathogenic strain Ab4 (GenBank accessionNo. AY665713) and a nonneuropathogenic strain V592 (GenBank accessionNo. AY464052) were extensively characterized and showed a nucleotidevariation rate of approximately 0.1% (Nugent et al., J. Virol 80,4047-4060, 2006). It was found that only a minority of EHV-1 strains arecapable of inducing neurological disorders, although all strains cancause respiratory disease and abortion (Mumford et al., J. Reprod FertilSuppl 35, 509-518, 1987; Ostlund, Vet Clin North Am Equine Pract 9,283-294, 1993; Wilson, Vet Clin North Am Equine Pract 13, 53-72, 1997).Recently, epidemiological as well as reverse-genetic studies have shownthat a single-nucleotide polymorphism at position 2254 (G/A2254) of openreading frame 30 (ORF30), encoding viral DNA polymerase (Pol), will leadto a variation at the amino acid position 752 (D/N752), which isassociated with the virus's neuropathogenic potential (Goodman et al., JBiol Chem 281, 18193-18200, 2007; Van de Walle et al., J. Infect Dis200, 20-25, 2009; Smith et al., Vet. Microbiol., 141, 5-11, 2010). Ithas been shown that residue 752 in the essential Pol of EHV-1 is notrequired for virus growth and that the N752 mutation confers adrug-sensitive phenotype to the virus (Ma et al., 2010).

Glycoprotein C (gC) of EHV-1 was shown to play important role in theearly steps of infection and in release of virions (Osterrieder, VirusResearch 2, 165, 1999). Glycoprotein C of EHV-1 is non-essential forvirus growth. It mediates primary attachment, and is required forefficient virus replication in primary equine cells (Osterrieder, 1999).

Conventional killed vaccines provide only partial clinical andvirological protection against respiratory infections, but do notprevent cell-associated viremia. Considering the susceptibility ofanimals, including humans, to herpesvirus, a means of preventingherpesvirus infection and protecting animals is essential. Accordingly,there is a need for an effective vaccine against herpesvirus.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention provides a composition or vaccine that contains arecombinant Equine Herpesvirus-1 (EHV-1). In particular, the presentinvention provides a recombinant EHV-1 that contains a mutatedGlycoprotein C (gC) gene that is non-functional. The gC gene of therecombinant EHV-1 may be deleted. The gC gene may encode a mutated gCprotein wherein the N-terminal region of the gC protein is deleted. Therecombinant EHV-1 may further comprise a DNA polymerase (Pol) geneencoding a Pol having an asparagine (N) at the amino acid position 752.The recombinant EHV-1 may comprise a DNA polymerase (Pol) gene encodinga Pol having an asparagine (N) at the amino acid position 752. The EHV-1may be EHV-1 RacL strain.

The invention provides methods for inducing an immunogenic or protectiveresponse against EHV-1, as well as methods for preventing EHV-1 ordisease state(s) caused by EHV-1, comprising administering thecomposition or vaccine of the present invention. The invention alsoprovides methods of vaccinating an animal comprising at least oneadministration of the composition or recombinant EHV-1.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

The following detailed description, given by way of example, and whichis not intended to limit the invention to specific embodimentsdescribed, may be understood in conjunction with the accompanyingfigures, incorporated herein by reference, in which:

FIG. 1 is the table showing the corresponding SEQ ID NO assigned topolynucleotide and protein sequences.

FIGS. 2A-2C depict the cloning scheme. FIG. 2D shows the amino acidmutation in EHV-1 polymerase.

FIGS. 3A-3B show the indirect immunofluorescence assay (IFA) of RK13-Poland RK13.

FIGS. 4A-4B depict the growth of L11-_ΔPol EYFP in RK13-Pol v. L11-_ΔPolEYFP in RK13.

FIGS. 5A-5F depict the IFA of RacL11, L11_D752NΔgC and L11_D752NΔgC revusing anti-EHV-1 gC MAb.

FIG. 6 depicts the in vitro growth of RacL11, L11_D752N, L11_D752NΔgCand L11_D752NΔgC rev measured by attachment assay.

FIG. 7 depicts the in vitro growth of RacL11, L11_D752N, L11_D752NΔgCand L11_D752NΔgC rev determined by comparing plaque sizes.

FIG. 8 depicts the growth kinetics of RacL11, L11_D752N, L11_D752NΔgCand L11_D752NΔgC rev by extracellular and intracellular titers.

FIGS. 9A and 9B depict the mean virus titers in lungs infected withRacL11, L11_D752N, L11_D752NΔgC, L11_D752N ΔgC rev 2 and PBS (control) 2and 4 days post infection (p.i.), 2 and 4 days post challenge (p.c.).

FIG. 10 depicts the histopathological changes of the lungs of miceinfected with RacL11, L11_D752N, L11_D752NΔgC, L11_D752N ΔgC rev on day2 p.i.

FIGS. 11A-11B depict the serology against EHV-1 using the complementfixation (CF) and virus neutralization (VN) tests.

FIG. 12 depicts the duration of viraemia in vaccinated horses.

FIG. 13 depicts the virus shedding in nasal swabs from the vaccinatedhorses.

FIGS. 14A-14C show the polynucleotide and protein sequence alignments ofEHV-1 DNA polymerase.

FIGS. 15A-15B show the polynucleotide and protein sequence alignments ofEHV-1 glycoprotein C.

FIG. 16 shows the DNA and protein sequences.

DETAILED DESCRIPTION

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to it in U.S. patent law; e.g., they canmean “includes”, “included”, “including”, and the like; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V. published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The word “or” means any one member of a particular list andalso includes any combination of members of that list.

The term “EHV-1 N strain” as used herein refers to any EHV-1 strainwhich has an asparagine (N) at the amino acid position 752 of its DNApolymerase. The EHV-1 N strain may be wild-type strain that comprises aDNA polymerase comprising an asparagine (N) at the amino acid position752. The EHV-1 N strain may be mutated or recombinant EHV-1 strainwherein the DNA polymerase is engineered to have an asparagine (N) atthe amino acid position 752.

The term “EHV-1 D strain” as used herein refers to any EHV-1 strainwhich has an Aspartic acid (D) at the amino acid position 752 of its DNApolymerase. The EHV-1 D strain may be a wild-type strain that comprisesa DNA polymerase comprising an aspartic acid (D) at the amino acidposition 752. The EHV-1 D strain may be a mutated or recombinant EHV-1strain wherein the DNA polymerase is engineered to have an aspartic acid(D) at the amino acid position 752.

By “animal” is intended mammals, human, birds, and the like. The animalmay be selected from the group consisting of equine (e.g., horse, zebra,donkey), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline(e.g., lions, tigers, domestic cats, wild cats, other big cats, andother feline including cheetahs and lynx), ovine (e.g., sheep), bovine(e.g., cattle, cow, buffalo), swine (pig), avian (e.g., chicken, duck,goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich,emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon,ape), and fish. The term “animal” also includes an individual animal inall stages of development, including embryonic and fetal stages.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of consecutive amino acid residues.

The term “nucleic acid”, “nucleotide”, and “polynucleotide” refers toRNA or DNA and derivatives thereof, such as those containing modifiedbackbones. It should be appreciated that the invention providespolynucleotides comprising sequences complementary to those describedherein. Polynucleotides according to the invention can be prepared indifferent ways (e.g. by chemical synthesis, by gene cloning etc.) andcan take various forms (e.g. linear or branched, single or doublestranded, or a hybrid thereof, primers, probes etc.).

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genes orpolynucleotides include introns and exons as in genomic sequence, orjust the coding sequences as in cDNAs, such as an open reading frame(ORF), starting from the start codon (methionine codon) and ending witha termination signal (stop codon). Genes and polynucleotides can alsoinclude regions that regulate their expression, such as transcriptioninitiation, translation and transcription termination. Thus, alsoincluded are promoters and ribosome binding regions (in general theseregulatory elements lie approximately between 60 and 250 nucleotidesupstream of the start codon of the coding sequence or gene; Doree S M etal.; Pandher K et al.; Chung J Y et al.), transcription terminators (ingeneral the terminator is located within approximately 50 nucleotidesdownstream of the stop codon of the coding sequence or gene; Ward C K etal.). Gene or polynucleotide also refers to a nucleic acid fragment thatexpresses mRNA or functional RNA, or encodes a specific protein, andwhich includes regulatory sequences.

As used herein, the term “antigen” or “immunogen” means a substance thatinduces a specific immune response in a host animal. The antigen maycomprise a whole organism, killed, attenuated or live; a subunit orportion of an organism; a recombinant vector containing an insertexpressing an epitope, polypeptide, peptide, protein, or fragmentthereof with immunogenic properties; a piece or fragment of nucleic acidcapable of inducing an immune response upon presentation to a hostanimal; a protein, a polypeptide, a peptide, an epitope, a hapten, orany combination thereof. Alternately, the immunogen or antigen maycomprise a toxin or antitoxin.

By definition, an epitope is an antigenic determinant that isimmunologically active in the sense that once administered to the host,it is able to evoke an immune response of the humoral (B cells) and/orcellular type (T cells). These are particular chemical groups or peptidesequences on a molecule that are antigenic. An antibody specificallybinds a particular antigenic epitope on a polypeptide. Specific,non-limiting examples of an epitope include a tetra- to penta-peptidesequence in a polypeptide, a tri- to penta-glycoside sequence in apolysaccharide. In the animal most antigens will present several or evenmany antigenic determinants simultaneously. Such a polypeptide may alsobe qualified as an immunogenic polypeptide and the epitope may beidentified as described further.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “purified” as used herein does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedpolypeptide preparation is one in which the polypeptide is more enrichedthan the polypeptide is in its natural environment. A polypeptidepreparation is substantially purified such that the polypeptiderepresents several embodiments at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, or at least 98%, of the total polypeptidecontent of the preparation. The same applies to polynucleotides. Thepolypeptides disclosed herein can be purified by any of the means knownin the art.

The present invention provides a composition or vaccine comprising arecombinant viral vector Equine Herpesvirus-1 (EHV-1). In one aspect,the present invention provides a recombinant EHV-1 that comprises amutated Glycoprotein C (gC) gene. In another aspect, the presentinvention provides a recombinant EHV-1 that comprises a DNA polymerasehaving an asparagine (N) at the amino acid position 752. In yet anotheraspect, the present invention provides a recombinant EHV-1 wherein theEHV-1 is an EHV-1 N strain. The recombinant EHV-1 N strain may comprisea mutated gC gene. In yet another aspect, the recombinant EHV-1comprises a mutated gC gene and a DNA polymerase having an asparagine(N) at the amino acid position 752. The composition or vaccine of thepresent invention may further comprise a pharmaceutically or veterinaryacceptable vehicle, diluent, adjuvant, or excipient.

The term “composition” comprises any vaccine or immunologicalcomposition, once it has been injected to a host, including canines,felines, equine and humans, that induces an immune response in the host,and/or protects the host from leukemia, and/or which may preventimplantation of the parasite, and/or which may prevent diseaseprogression in infected subjects, and/or which may limit the diffusionof runaway parasites to internal organs. This may be accomplished uponvaccination according to the present invention through the induction ofcytokine secretion, notably IFN-gamma secretion (as example of a methodof measurement of IFN-gamma secretion, the Quantikine® immunoassay fromR&D Systems Inc. (catalog number#CAIF00) could be used (Djoba Siawaya JF et al.)).

The present invention provides a recombinant EHV-1 comprising a DNApolymerase having an asparagine (N) at the amino acid position 752.Homologs of polypeptides of EHV-1 DNA polymerase are intended to bewithin the scope of the present invention. As used herein, the term“homologs” includes orthologs, analogs and paralogs. The tem “anologs”refers to two polynucleotides or polypeptides that have the same orsimilar function, but that have evolved separately in unrelatedorganisms. The term “orthologs” refers to two polynucleotides orpolypeptides from different species, but that have evolved from a commonancestral gene by speciation. Normally, orthologs encode polypeptideshaving the same or similar functions. The term “paralogs” refers to twopolynucleotides or polypeptides that are related by duplication within agenome. Paralogs usually have different functions, but these functionsmay be related. Analogs, orthologs, and paralogs of a wild-typepolypeptide can differ from the wild-type polypeptide bypost-translational modifications, by amino acid sequence differences, orby both. In particular, homologs of the invention will generally exhibitat least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequenceidentity, with all or part of the wild-type polypeptide orpolynucleotide sequences, and will exhibit a similar function.

In one aspect of the present invention, the recombinant EHV-1 comprisesan EHV-1 DNA polymerase comprising an asparagine (N) at the amino acidposition 752 or equivalent position of a polypeptide having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ IDNO: 2, 4, 6, 37, 13, 14, or 15. In another aspect, the present inventionprovides fragments and variants of the EHV-1 DNA polymerases, which mayreadily be prepared by one of skill in the art using well-knownmolecular biology techniques. Variants are homologous polypeptideshaving an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or99.9% identical to the amino acid sequence as set forth in SEQ ID NO: 2,4, 6, 37, 13, 14, or 15. Variants include allelic variants. The term“allelic variant” refers to a polynucleotide or a polypeptide containingpolymorphisms that lead to changes in the amino acid sequences of aprotein and that exist within a natural population (e.g., a virusspecies or variety). Such natural allelic variations can typicallyresult in 1-5% variance in a polynucleotide or a polypeptide. Allelicvariants can be identified by sequencing the nucleic acid sequence ofinterest in a number of different species, which can be readily carriedout by using hybridization probes to identify the same genetic locus inthose species. Any and all such nucleic acid variations and resultingamino acid polymorphisms or variations that are the result of naturalallelic variation and that do not alter the functional activity of geneof interest, are intended to be within the scope of the invention.

As used herein, the term “derivative” or “variant” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has one ormore conservative amino acid variations or other minor modificationssuch that (1) the corresponding polypeptide has substantially equivalentfunction when compared to the wild type polypeptide or (2) an antibodyraised against the polypeptide is immunoreactive with the wild-typepolypeptide. These variants or derivatives include polypeptides havingminor modifications of the EHV-1 DNA polymerase primary amino acidsequences that may result in peptides which have substantiallyequivalent activity as compared to the unmodified counterpartpolypeptide. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. The term “variant” furthercontemplates deletions, additions and substitutions to the sequence, solong as the polypeptide functions to produce an immunological responseas defined herein. The modifications may be any amino acid change atamino acid positions other than position 752 of SEQ ID NO: 2, 4, 6, 37,13, 14, or 15.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

Procedures to determine fragments of polypeptide and epitope such as,generating overlapping peptide libraries (Hemmer B. et al.), Pepscan(Geysen H. M. et al., 1984; Geysen H. M. et al., 1985; Van der Zee R. etal.; Geysen H. M.) and algorithms (De Groot A. et al.; Hoop T. et al.;Parker K. et al.), can be used in the practice of the invention, withoutundue experimentation. Generally, antibodies specifically bind aparticular antigenic epitope. Specific, non-limiting examples ofepitopes include a tetra- to penta-peptide sequence in a polypeptide, atri- to penta glycoside sequence in a polysaccharide. In animals mostantigens will present several or even many antigenic determinantssimultaneously. Preferably wherein the epitope is a protein fragment ofa larger molecule it will have substantially the same immunologicalactivity as the total protein.

In one aspect, the present invention provides a polynucleotide encodingan EHV-1 DNA polymerase comprising an asparagine (N) at the amino acidposition 752 or an equivalent position of a polypeptide having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ IDNO: 2, 4, 6, 37, 13, 14, or 15, or a conservative variant, an allelicvariant, a homolog or an immunogenic fragment comprising at least eightor at east ten consecutive amino acids of one of these polypeptides, ora combination of these polypeptides.

In another aspect, the present invention provides a polynucleotidehaving a nucleotide sequence as set forth in SEQ ID NO: 1, 3, 5, or 36,or a variant thereof. In yet another aspect, the present inventionprovides a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 1, 3, 5 or 36, ora variant thereof.

The polynucleotides of the disclosure include sequences that aredegenerate as a result of the genetic code, e.g., optimized codon usagefor a specific host. As used herein, “optimized” refers to apolynucleotide that is genetically engineered to increase its expressionin a given species. To provide optimized polynucleotides coding for anEHV-1 DNA polymerase, the DNA sequence of the EHV-1 DNA polymerase genecan be modified to 1) comprise codons preferred by highly expressedgenes in a particular species; 2) comprise an A+T or G+C content innucleotide base composition to that substantially found in said species;3) form an initiation sequence of said species; or 4) eliminatesequences that cause destabilization, inappropriate polyadenylation,degradation and termination of RNA, or that form secondary structurehairpins or RNA splice sites. Increased expression of EHV-1 DNApolymerase in said species can be achieved by utilizing the distributionfrequency of codon usage in eukaryotes and prokaryotes, or in aparticular species. The term “frequency of preferred codon usage” refersto the preference exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. There are 20 naturalamino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included in thedisclosure as long as the amino acid sequence of the EHV-1 DNApolymerase encoded by the nucleotide sequence is functionally unchanged.

In one aspect, the present invention provides a recombinant EHV-1 thatcontains a mutated Glycoprotein C (gC) gene. The term “mutated gC gene”refers to the gC gene of EHV-1 that is altered or engineered whichresults in a non-functional gC protein upon expression. The alterationor engineering of the gC gene includes mutation or deletion of a segmentof the gC gene which is essential for the expression of a functional gCprotein. The deletion of the gC gene may be a deletion of thepolynucleotides encoding the amino-terminal region of the gC protein.The deleted amino-terminal regions of the gC protein may be any length,for example, a region of 1-10 amino acids, or 11-20 amino acids, or21-30 amino acids, or 31-40 amino acids, or 41-60 amino acids, or 61-90amino acids, or 91-120 amino acids, or 121-160 amino acids. The term“mutated gC gene” also includes deletion of the entire gC gene of EHV-1wherein gC protein is not expressed.

In one aspect, the present invention provides a recombinant EHV-1wherein the Glycoprotein C (gC) gene in the native (wild-type) EHV-1genome encoding the gC protein is deleted. The term “Glycoprotein C (gC)gene” includes any gene or polynucleotide that encodes the GlycoproteinC (gC) of EHV-1, and homologs, fragments or variants thereof. The gCgene may encode a gC protein having at least 75%, 80%, 85%, 90%, 95%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 8, 10, or 12, or avariant thereof. The gC gene having at least 75%, 80%, 85%, 90%, 95%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO:7, 9, or 11 is alsoencompassed in the present invention. In another aspect, the presentinvention provides a recombinant EHV-1 wherein the Glycoprotein C (gC)gene in the native (wild-type) EHV-1 genome encoding the gC protein isaltered or engineered resulting in a mutated gC protein. In yet anotheraspect, the engineered gC gene encodes a mutated gC protein wherein theN-terminal region of the gC protein is deleted.

The sequence identity between two amino acid sequences may beestablished by the NCBI (National Center for Biotechnology Information)pairwise blast and the blosum62 matrix, using the standard parameters(see, e.g., the BLAST or BLASTX algorithm available on the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA)server), as well as in Altschul et al.; and thus, this document speaksof using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by theterm “blasts”.

Alternatively or additionally, the term “identity”, for instance, withrespect to a nucleotide or amino acid sequence, may indicate aquantitative measure of homology between two sequences. The percentsequence homology may be calculated as: (N_(ref)−N_(dif))*100/N_(ref),wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned and wherein N_(ref) is the number of residues inone of the sequences. Hence, the DNA sequence AGTCAGTC will have asequence identity of 75% with the sequence AATCAATC (N_(ref)=8;N_(dif)=2).

Alternatively or additionally, “identity” with respect to sequences canrefer to the number of positions with identical nucleotides or aminoacids divided by the number of nucleotides or amino acids in the shorterof the two sequences wherein alignment of the two sequences can bedetermined in accordance with the Wilbur and Lipman algorithm (Wilburand Lipman), for instance, using a window size of 20 nucleotides, a wordlength of 4 nucleotides, and a gap penalty of 4, and computer-assistedanalysis and interpretation of the sequence data including alignment canbe conveniently performed using commercially available programs (e.g.,Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences aresaid to be similar, or have a degree of sequence identity or homologywith DNA sequences, thymidine (T) in the DNA sequence is consideredequal to uracil (U) in the RNA sequence. Thus, RNA sequences are withinthe scope of the invention and can be derived from DNA sequences, bythymidine (T) in the DNA sequence being considered equal to uracil (U)in RNA sequences.

The sequence identity or sequence similarity of two amino acidsequences, or the sequence identity between two nucleotide sequences canbe determined using Vector NTI software package (Invitrogen, 1600Faraday Ave., Carlsbad, Calif.).

Recombinant vectors disclosed herein may include a polynucleotideencoding a polypeptide, a variant thereof or a fragment thereof.Recombinant vectors may include plasmids and viral vectors and may beused for in vitro or in vivo expression. Recombinant vectors may includefurther a signal peptide. Signal peptides are short peptide chain (3-60amino acids long) that direct the post-translational transport of aprotein (which are synthesized in the cytosol) to certain organellessuch as the nucleus, mitochondrial matrix, endoplasmic reticulum,chloroplast, apoplast and peroxisome. Typically, the naturally occurringEHV-1 proteins may be translated as precursors, having an N-terminalsignal peptide sequence and a “mature” protein domain. The signalpeptide may be cleaved EHV-1 protein or a peptide signal from a secretedprotein e.g. the signal peptide from the tissue plasminogen activatorprotein (tPA), in particular the human tPA (S. Friezner Degen et al.; R.Rickles et al.; D. Berg. et al.), or the signal peptide from theInsulin-like growth factor 1 (IGF1), in particular the equine IGF1 (K.Otte et al.), the canine IGF1 (P. Delafontaine et al.), the feline IGF1(WO03/022886), the bovine IGF1 (S. Lien et al.), the porcine IGF1 (M.Muller et al.), the chicken IGF1 (Y. Kajimoto et al.), the turkey IGF1(GenBank accession number AF074980). The signal peptide from IGF1 may benatural or optimized which may be achieved by removing cryptic splicesites and/or by adapting the codon usage. Upon translation, theunprocessed polypeptide may be cleaved at a cleavage site to lead to themature polypeptide. The cleavage site may be predicted using the methodof Von Heijne (1986).

A plasmid may include a DNA transcription unit, for instance a nucleicacid sequence that permits it to replicate in a host cell, such as anorigin of replication (prokaryotic or eukaryotic). A plasmid may alsoinclude one or more selectable marker genes and other genetic elementsknown in the art. Circular and linear forms of plasmids are encompassedin the present disclosure.

In a further aspect, the present invention relates to an in vivoexpression vector comprising a polynucleotide sequence, which containsand expresses in vivo in a host the EHV-1 antigen, polypeptides and/orvariants or fragments thereof.

The in vivo expression vector may include any transcription unitcontaining a polynucleotide or a gene of interest and those essentialelements for its in vivo expression. These expression vectors may beplasmids or recombinant viral vectors. For in vivo expression, thepromoter may be of viral or cellular origin. In one embodiment, thepromoter may be the cytomegalovirus (CMV) early promoter (CMV-IEpromoter), the SV40 virus early or late promoter or the Rous Sarcomavirus LTR promoter, a promoter of a cytoskeleton gene, such as thedesmin promoter (Kwissa M. et al.), or the actin promoter (Miyazaki J.et al.). When several genes are present in the same plasmid, they may beprovided in the same transcription unit or in different units.

As used herein, the term “plasmid” may include any DNA transcriptionunit comprising a polynucleotide according to the invention and theelements necessary for its in vivo expression in a cell or cells of thedesired host or target; and, in this regard, it is noted that asupercoiled or non-supercoiled, circular plasmid, as well as a linearform, are intended to be within the scope of the invention. The plasmidsmay also comprise other transcription-regulating elements such as, forexample, stabilizing sequences of the intron type. In severalembodiments, the plasmids may include the first intron of CMV-IE (WO89/01036), the intron II of the rabbit beta-globin gene (van Ooyen etal.), the signal sequence of the protein encoded by the tissueplasminogen activator (tPA; Montgomery et al.), and/or a polyadenylationsignal (polyA), in particular the polyA of the bovine growth hormone(bGH) gene (U.S. Pat. No. 5,122,458) or the polyA of the rabbitbeta-globin gene or of SV40 virus.

The pharmaceutically acceptable vehicles or diluents or excipients oradjuvants of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the polypeptides, plasmids, viral vectors herein disclosed.In general, the nature of the vehicle or excipient will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. For solid compositions (for example,freeze-dried pastille, powder, pill, tablet, or capsule forms),conventional non-toxic solid vehicles or diluents or excipients oradjuvants can include, for example, pharmaceutical grades of mannitol,lactose, starch, or magnesium stearate. In addition to biologicallyneutral vehicles or diluents or excipients or adjuvants, immunogeniccompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

The compositions or vaccines according to the instant invention mayinclude recombinant vectors encoding any polypeptide or antigenaccording to the present invention as described above.

Multiple insertions may be done in the same vector using differentinsertion sites or using the same insertion site. When the sameinsertion site is used, each polynucleotide insert, which may be anypolynucleotide of the present invention aforementioned, may be insertedunder the control of the same and/or different promoters. The insertioncan be done tail-to-tail, head-to-head, tail-to-head, or head-to-tail.IRES elements (Internal Ribosome Entry Site, see EP 0803573) can also beused to separate and to express multiple inserts operably linked to thesame and/or different promoters.

More generally, the present invention encompasses in vivo expressionvectors including any plasmid (EP-A2-1001025; Chaudhuri P.) containingand expressing in vivo in a host the polynucleotide or gene of EHV-1polypeptide, variant or fragment as described above and elementsnecessary for its in vivo expression.

In a specific, non-limiting example, the pVR1020 or pVR1012 plasmid(VICAL Inc.; Luke C. et al.; Hartikka J. et al.), pVR2001-TOPA (orpVR2001-TOPO) (Oliveira F. et al.) or pAB110 (U.S. Pat. No. 6,852,705)can be utilized as a vector for the insertion of a polynucleotidesequence. The pVR1020 plasmid is derived from pVR1012 and contains thehuman tPA signal sequence. The pVR1020 is a plasmid backbone availablefrom Vical, Inc., (San Diego, Calif.) which has been previously used,see, e.g., U.S. Pat. Nos. 6,451,769 and 7,078,507. As described inOliveira et al., plasmid pVR2001-TOPO (or pVR2001-TOPA) is pVR1020modified by the addition of topoisomerases flanking the cloning site andcontaining coding for and expressing a signal secretory peptide, forexample, tissue plasminogen activator signal peptide (tPA), thatincreases the likelihood of producing a secreted protein (Oliveira F. etal.).

Each plasmid may comprise or contain or consist essentially of, thepolynucleotide according to the present invention, operably linked to apromoter or under the control of a promoter or dependent upon apromoter, wherein the promoter may be advantageously adjacent to thepolynucleotide for which expression is desired. In general, it isadvantageous to employ a strong promoter that is functional ineukaryotic cells. One example of a useful promoter may be the immediateearly cytomegalovirus promoter (CMV-IE) of human or murine origin, or itmay optionally have another origin such as from rat or guinea pig. TheCMV-IE promoter may comprise the actual promoter part, which may or maynot be associated with the enhancer part. Reference can be made to EP260 148, EP 323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615,as well as to WO 87/03905. The CMV-IE promoter may advantageously be ahuman CMV-IE (Boshart M. et al.) or murine CMV-IE. In more generalterms, the promoter may have either a viral or a cellular origin. Astrong viral promoter other than CMV-IE that may be usefully employed inthe practice of the invention is the early/late promoter of the SV40virus or the LTR promoter of the Rous sarcoma virus. A strong cellularpromoter that may be usefully employed in the practice of the inventionis the promoter of a gene of the cytoskeleton, such as the desminpromoter (Kwissa M. et al.), or the actin promoter (Miyazaki J. et al.).Functional sub fragments of these promoters, i.e., portions of thesepromoters that maintain adequate promoter activity, are included withinthe present invention, e.g. truncated CMV-IE promoters according to WO98/00166 or U.S. Pat. No. 6,156,567 and may be used in the practice ofthe invention. A promoter useful in the practice of the inventionconsequently may include derivatives and/or sub fragments of afull-length promoter that maintain adequate promoter activity and hencefunction as a promoter, and which may advantageously have promoteractivity that is substantially similar to that of the actual orfull-length promoter from which the derivative or sub fragment isderived, e.g., akin to the activity of the truncated CMV-IE promoters ofU.S. Pat. No. 6,156,567 in comparison to the activity of full-lengthCMV-IE promoters. Thus, a CMV-IE promoter in the practice of theinvention may comprise or consist essentially of or consist of thepromoter portion of the full-length promoter and/or the enhancer portionof the full-length promoter, as well as derivatives and/or sub fragmentsthereof.

Advantageously, the plasmids comprise or consist essentially of otherexpression control elements. It is especially advantageous toincorporate stabilizing sequence(s), e.g., intron sequence(s), forexample, the first intron of the hCMV-IE (WO 89/01036), the intron II ofthe rabbit β-globin gene (van Ooyen et al.). As to the polyadenylationsignal (polyA) for the plasmids and viral vectors other than poxviruses,use can be made of the poly(A) signal of the bovine growth hormone (bGH)gene (see U.S. Pat. No. 5,122,458), or the poly(A) signal of the rabbitβ-globin gene or the poly(A) signal of the SV40 virus.

More generally, the present invention encompasses in vivo expressionvectors including any recombinant viral vector containing apolynucleotide or gene encoding one or more EHV-1 polypeptide, variantsor fragments as described above, including any elements necessary forits in vivo expression.

The recombinant viral vector may be a Herpesvirus, such as an equineHerpesvirus-1 (EHV-1) as described above. The EHV-1 vector may bederived from the RacH strain, the RacL strain, the Ab4 strain, the V592strain, the Kentucky D strain (TACC No. VR-700), the 438/77 strain (ATCCNo. VR-2229), the AB69 strain (ATCC No. VR-2581), the EHV-1 NY03 strain,or a combination of EHV-1 RacH or RacL strains. In one embodiment thepolynucleotide to be expressed is inserted under the control of apromoter functional in eukaryotic cells, advantageously a CMV-IEpromoter (murine or human). A poly(A) sequence and terminator sequencecan be inserted downstream the polynucleotide to be expressed, e.g.bovine growth hormone or a rabbit β-globin gene polyadenylation signal.

In one embodiment, the viral vector could be Newcastle Disease Virus(US2012/052089). In another embodiment, the vial vector could beselected from, for example, the poxviruses, especially avipox viruses,such as fowlpox viruses or canarypox viruses. In one embodiment, thefowlpox virus is a TROVAC (see WO 96/40241). In another embodiment, thecanarypox vector is an ALVAC. The use of these recombinant viral vectorsand the insertion of polynucleotides or genes of interest are fullydescribed in U.S. Pat. No. 5,174,993; U.S. Pat. No. 5,505,941 and U.S.Pat. No. 5,766,599 for fowlpox, and in U.S. Pat. No. 5,756,103 forcanarypox. More than one insertion site inside the viral genome could beused for the insertion of multiple genes of interest.

For recombinant vectors based on a poxvirus vector, a vaccinia virus oran attenuated vaccinia virus, (for instance, MVA, a modified Ankarastrain obtained after more than 570 passages of the Ankara vaccinestrain on chicken embryo fibroblasts; see Stickl & Hochstein-Mintzel;Sutter et al.; available as ATCC VR-1508; or NYVAC, see U.S. Pat. No.5,494,807, and U.S. Pat. No. 5,494,807 which discuss the construction ofNYVAC, as well as variations of NYVAC with additional ORFs deleted fromthe Copenhagen strain vaccinia virus genome, as well as the insertion ofheterologous coding nucleic acid molecules into sites of thisrecombinant, and also, the use of matched promoters; see also WO96/40241), an avipox virus or an attenuated avipox virus (e.g.,canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC; see,e.g., U.S. Pat. Nos. 5,505,941, 5,494,807) can be used. Attenuatedcanarypox viruses are described in U.S. Pat. No. 5,756,103 (ALVAC) andWO 01/05934. Reference is also made to U.S. Pat. No. 5,766,599 whichpertains to the attenuated fowlpox strain TROVAC. Reference is made tothe canarypox available from the ATCC under access number VR-111.Numerous fowlpox virus vaccination strains are also available, e.g. theDIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIOLE vaccinemarketed by INTERVET. For information on the method used to generaterecombinants thereof and how to administer recombinants thereof, theskilled artisan can refer documents cited herein and to WO 90/12882,e.g., as to vaccinia virus, mention is made of U.S. Pat. Nos. 4,769,330,4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia; asto fowlpox, mention is made of U.S. Pat. Nos. 5,174,993, 5,505,941 and5,766,599 inter alia; as to canarypox, mention is made of U.S. Pat. No.5,756,103 inter alia. When the expression vector is a vaccinia virus,insertion site or sites for the polynucleotide or polynucleotides to beexpressed are advantageously at the thymidine kinase (TK) gene orinsertion site, the hemagglutinin (HA) gene or insertion site, theregion encoding the inclusion body of the A type (ATI). In the case ofcanarypox, advantageously the insertion site or sites are ORF(s) C3, C5and/or C6. In the case of fowlpox, advantageously the insertion site orsites are ORFs F7 and/or F8. The insertion site or sites for MVA virusare advantageously as in various publications, including Carroll M. W.et al.; Stittelaar K. J. et al.; Sutter G. et al.; and, in this regardit is also noted that the complete MVA genome is described in AntoineG., Virology, which enables the skilled artisan to use other insertionsites or other promoters. Advantageously, the polynucleotide to beexpressed is inserted under the control of a specific poxvirus promoter,e.g., the vaccinia promoter 7.5 kDa (Cochran et al.), the vacciniapromoter I3L (Riviere et al.), the vaccinia promoter HA (Shida), thecowpox promoter ATI (Funahashi et al.), the vaccinia promoter H6 (TaylorJ. et al.; Guo P. et al. J.; Perkus M. et al.).

Any of the polynucleotides disclosed here may be expressed in vitro byDNA transfer or expression vectors into a suitable host cell. The hostcell may be prokaryotic or eukaryotic. The term “host cell” alsoincludes any progeny of the subject host cell. Methods of stabletransfer, meaning that the foreign polynucleotide is continuouslymaintained in the host cell, are known in the art. Host cells mayinclude bacteria (for example, Escherichia coli), yeast, insect cells,and vertebrate cells. Methods of expressing DNA sequences in eukaryoticcells are well known in the art. As a method for in vitro expression,recombinant Baculovirus vectors (for example, Autographa CaliforniaNuclear Polyhedrosis Virus (AcNPV)) may be used with the nucleic acidsdisclosed herein. For example, polyhedrin promoters may be utilized withinsect cells (for example, Spodoptera frugiperda cells, like Sf9 cellsavailable at the ATCC under the Accession number CRL 1711, or Sf21cells) (see for example, Smith et al.; Pennock et al.; Vialard et al.;Verne A.; O'Reilly et al.; Kidd I. M. & Emery V. C.; EP 0370573; EP0265785; U.S. Pat. No. 4,745,051). For expression, the BaculoGoldStarter Package (Cat #21001K) from Pharmingen (Becton Dickinson) may beused. As a method for in vitro expression, recombinant E. coli may beused with a vector. For example, when cloning in bacterial systems,inducible promoters such as arabinose promoter, pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter), and the like may beused. Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl2method using procedures well known in the art. Alternatively, MgCl2 orRbCl can be used. Transformation can also be performed byelectroporation. When the host is a eukaryote, such methods oftransduction of DNA as calcium phosphate coprecipitates, conventionalmechanical procedures such as microinjection, electroporation, insertionof a plasmid encased in liposomes, or virus vectors may be used.Eukaryotic cells may also be cotransformed with L. longipalpispolynucleotide sequences, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector (see above), such asa herpes virus or adenovirus (for example, canine adenovirus 2), totransiently transduce eukaryotic cells and express the protein (GluzmanE A). In addition, a transfection agent can be utilized, such asdioleoyl-phosphatidyl-ethanolamme (DOPE).

Isolation and purification of recombinantly expressed polypeptide may becarried out by conventional means including preparative chromatography(for example, size exclusion, ion exchange, affinity), selectiveprecipitation and ultra-filtration. Examples of state of the arttechniques that can be used, but not limited to, may be found in“Protein Purification Applications”, Second Edition, edited by Simon Roeand available at Oxford University Press. Such a recombinantly expressedpolypeptide is part of the present disclosure. The methods forproduction of any polypeptide according to the present invention asdescribed above are also encompassed, in particular the use of arecombinant expression vector comprising a polynucleotide according tothe disclosure and of a host cell.

The vaccines containing recombinant viral vectors according to theinvention may be freeze-dried, advantageously with a stabilizer.Freeze-drying can be done according to well-known standard freeze-dryingprocedures. The pharmaceutically or veterinary acceptable stabilizersmay be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose,glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al.;Israeli E et al.), proteins such as peptone, albumin, lactalbumin orcasein, protein containing agents such as skimmed milk (Mills C K etal.; Wolff E et al.), and buffers (e.g. phosphate buffer, alkaline metalphosphate buffer). An adjuvant may be used to make soluble thefreeze-dried preparations.

Any vaccine composition according to the invention can alsoadvantageously contain one or more adjuvant.

The plasmid-based vaccines may be formulated with cationic lipids,advantageously with DMRIE(N-(2-hydroxyéthyl)-N,N-diméthyl-2,3-bis(tetradécyloxy)-1-propanammonium;WO96/34109), and advantageously in association with a neutral lipid, forexample DOPE (dioleoyl-phosphatidyl-ethanolamine; Behr J. P.), in orderto form DMRIE-DOPE. In one embodiment, the mixture is madeextemporaneously, and before its administration it is advantageous towait about 10 min to about 60 min, for example, about 30 min, for theappropriate mixture. When DOPE is used, the molar ratio of DMRIE/DOPEcan be from 95/5 to 5/95 and is advantageously 1/1. The weight ratioplasmid/DMRIE or DMRIE-DOPE adjuvant is, for example, from 50/1 to 1/10,from 10/1 to 1/5 or from 1/1 to 1/2.

Optionally a cytokine may be added to the composition, especially GM-CSFor cytokines inducing Th1 (e.g. IL12). These cytokines can be added tothe composition as a plasmid encoding the cytokine protein. In oneembodiment, the cytokines are from canine origin, e.g. canine GM-CSFwhich gene sequence has been deposited at the GenBank database(accession number S49738). This sequence can be used to create saidplasmid in a manner similar to what was made in WO 00/77210.

The recombinant viral vector-based vaccine may be combined with fMLP(N-formyl-methionyl-leucyl-phenylalanine; U.S. Pat. No. 6,017,537)and/or Carbomer adjuvant (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462, whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, advantageously not more than8, the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Forexample, the radicals are those containing from 2 to 4 carbon atoms,e.g. vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name CARBOPOL® (BF Goodrich, Ohio,USA) are appropriate. The products are cross-linked with an allylsucrose or with allyl pentaerythritol. Among them, there may beadvantageously mentioned CARBOPOL® 974P, 934P and 971P.

Among the copolymers of maleic anhydride and alkenyl derivative, thecopolymers

EMA® (Monsanto) which are copolymers of maleic anhydride and ethylene,linear or cross-linked, for example cross-linked with divinyl ether, areadvantageous. Reference may be made to J. Fields et al.

The polymers of acrylic or methacrylic acid and the copolymers EMA® areformed, for example, of basic units of the following formula in which:

-   -   R₁ and R₂, which are identical or different, represent H or CH₃    -   x=0 or 1, preferably x=1    -   y=1 or 2, with x+y=2

For the copolymers EMA®, x=0 and y=2. For the carbomers, x=y=1.

The dissolution of these polymers in water leads to an acid solution,which is neutralized, advantageously to physiological pH, in order toprovide the adjuvant solution into which the vaccine itself isincorporated. The carboxyl groups of the polymer are then partly in COO⁻form.

In one embodiment, a solution of adjuvant, especially of carbomer(Pharmeuropa, vol. 8, No. 2, June 1996), is prepared in distilled water,advantageously in the presence of sodium chloride, the solution obtainedbeing at an acidic pH. This stock solution is diluted by adding it tothe desired quantity (for obtaining the desired final concentration), ora substantial part thereof, of water charged with NaCl, advantageouslyphysiological saline (NaCl 9 g/l) all at once in several portions withconcomitant or subsequent neutralization (pH 7.3 to 7.4), advantageouslywith NaOH. This solution at physiological pH is used for mixing with thevaccine, which may be especially stored in freeze-dried, liquid orfrozen form.

The polymer concentration in the final vaccine composition can be from0.01% to 2% w/v, from 0.06 to 1% w/v, or from 0.1 to 0.6% w/v.

The subunit vaccine may be combined with adjuvants, like oil-in-water,water-in-oil-in-water emulsions based on mineral oil and/or vegetableoil and non ionic surfactants such as block copolymers, TWEEN®, SPAN®.Such emulsions are notably those described in page 147 of “VaccineDesign—The Subunit and Adjuvant Approach”, Pharmaceutical Biotechnology,1995, or TS emulsions, notably the TS6 emulsion, and LF emulsions,notably LF2 emulsion (for both TS and LF emulsions, see WO 04/024027).Other suitable adjuvants are for example vitamin E, saponins, andCARBOPOL® (Noveon; see WO 99/51269; WO 99/44633), aluminium hydroxide oraluminium phosphate (“Vaccine Design, The subunit and adjuvantapproach”, Pharmaceutical Biotechnology, vol. 6, 1995), biologicaladjuvants (i.e. C4b, notably murine C4b (Ogata R T et al.) or equineC4b, GM-CSF, notably equine GM-CSF (U.S. Pat. No. 6,645,740)), toxins(i.e. cholera toxins CTA or CTB, Escherichia coli heat-labile toxins LTAor LTB (Olsen C W et al.; Fingerut E et al.; Zurbriggen R et al.Peppoloni S et al.), and CpG (i.e. CpG #2395 (see Jurk M et al.), CpG#2142 (see SEQ. ID. NO: 890 in EP 1,221,955).

The composition or vaccine may also be associated with at least oneEHV-1 antigen, for example inactivated EHV-1. In a particularembodiment, the EHV-1 strain may be the RacH strain, the RacL strain,the Ab4 strain, the V592 strain, the Kentucky D strain (TACC No.VR-700), the 438/77 strain (ATCC No. VR-2229), the AB69 strain (ATCC No.VR-2581), EHV-1 NY03, or a combination of EHV-1 RacH or RacL strains.These strains of EHV-1 may be inactivated by chemical or physicalmethods. The chemical methods are notably BPL, formaldehyde. Thephysical methods may notably be sonication. The inactivated EHV-1vaccine may be combined with adjuvants, like those described previouslyfor subunit vaccines.

Another aspect of the present invention relates to methods ofvaccinating a host against EHV-1 using the vaccines or compositionsdisclosed herein.

The host may be any animals. In one embodiment, the host is an equine.

The routes of administration may be, for example, intramuscular (IM) orintradermal (ID) or transdermal (TD) or subcutaneous (SC). The means ofadministration may be, for example, a syringe with a needle, or needlefree apparatus, or a syringe with a needle coupled to electrotransfer(ET) treatment, or needle free apparatus coupled to ET treatment.

For plasmid-based vaccines, advantageous routes of administration may beID or IM. This administration may be through use of a syringe with aneedle or with a needle free apparatus like Dermojet or Biojector(Bioject, Oregon, USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.),see US 2006/0034867. The dosage may be from 50 μg to 500 μg per plasmid.When DMRIE-DOPE is added, 100 μg per plasmid may be utilized. WhenGM-CSF or other cytokines are used, the plasmid encoding this proteinmay be present at a dosage of from about 200 μg to about 500 μg and maybe 200 μg. The volume of doses can be between 0.01 ml and 0.5 ml, forexample, 0.25 ml. Administration may be provided with multiple points ofinjection.

Alternatively, plasmid-based vaccines may be administered via the IMroute coupled to electrotransfer (ET) treatment. The ET treatment may beperformed using an apparatus for electrotransfer and the specificationsof the manufacturer (i.e. Sphergen G250 generator (Sphergen SARL, EvryGenopole, France); MedPulser® DNA electroporation system (InnovioBiomedical Corporation, San Diego, Calif., USA)).

For recombinant viral vector-based vaccines, the routes ofadministration may advantageously be SC, IM, TD, or ID. Thisadministration may be made by a syringe with a needle or with a needlefree apparatus like Dermojet or Biojector (Bioject, Oregon, USA) orVetjet™ (Merial) or Vitajet™ (Bioject Inc.). The dosage may be fromabout 10⁴ pfu to about 10⁹ pfu per recombinant EHV vector. The volume ofdoses may be from about 0.01 ml to 0.2 ml, and is advantageously 0.1 ml.Administration may comprise multiple points of injection.

For the IM route the volume of the vaccine provided may be from 0.2 to 2ml, in particular from about 0.5 to 1 ml. The same dosages are utilizedfor any of the vectors of the present invention.

For subunit vaccines, the route of administration may advantageously bevia SC or IM or TD or ID. This administration may be made by a syringewith a needle or with a needle free apparatus like Dermojet or Biojector(Bioject, Oregon, USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.).The dosage may be from about 50 to about 500 μg, in particular fromabout 50 to about 150 μg, and more particularly from about 50 to about100 μg. The volume of the sub-unit vaccine provided is from 0.2 to 2 ml,in particular from about 0.5 to 1 ml.

In one aspect, the present invention relates to a vaccine strategy,which is based on a prime-boost administration regimen, where theprime-administration and the boost-administration utilize a compositioncomprising a pharmaceutically or veterinary acceptable excipient,diluent, adjuvant, or vehicle and the recombinant EHV-1 of the presentinvention. In another aspect, the present invention relates to a methodof vaccinating a subject or host susceptible to EHV-1 comprising aprime-boost administration regime.

A prime-boost regimen comprises at least one prime-administration and atleast one boost administration using at least one common antigen and/orvariants or fragments thereof. The vaccine used in prime-administrationmay be different in nature from those used as a later booster vaccine.It is further noted that both the prime-administration and theboost-administration may comprise the recombinant EHV-1 of the presentinvention. The prime-administration may comprise one or moreadministrations. Similarly, the boost-administration may comprise one ormore administrations.

The routes of administration, doses and volumes are as previouslydisclosed herein.

The prime-boost administrations may be carried out 2 to 6 weeks apart,for example, about 4 weeks apart. According to one embodiment, asemi-annual booster or an annual booster is also envisaged.

In one embodiment, the prime-boost administration regimen comprises atleast one prime-administration of a recombinant EHV-1 vaccine orcomposition of the present invention and at least oneboost-administration of an inactivated viral vaccine comprising theEHV-1 antigen, or a plasmid-based vaccine expressing the EHV-1 antigen,or a subunit vaccine comprising the EHV-1 antigen, or a combinationthereof.

In another embodiment, the prime-boost administration regimen comprisesat least one prime-administration of an inactivated viral vaccinecomprising the EHV-1 antigen, or a plasmid-based vaccine expressing theEHV-1 antigen, or a subunit vaccine comprising the EHV-1 antigen, or acombination thereof and at least one boost-administration of arecombinant EHV-1 vaccine or composition of the present invention.

Another aspect of the present invention relates to a kit for prime-boostvaccination according to the present invention. The kit may comprise atleast two vials: a first vial containing a vaccine for theprime-vaccination according to the present invention, and a second vialcontaining a vaccine for the boost-vaccination according to the presentinvention. The kit may advantageously contain additional first or secondvials for additional prime-vaccinations or additionalboost-vaccinations.

In one embodiment, the kit may comprise two vials, one containing aplasmid-based vaccine for the prime-vaccination according to the presentinvention, the other vial containing a recombinant viral vector-basedvaccine for the boost-vaccination according to the present invention.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding descriptions, practice the present invention toits fullest extent. The following detailed examples are to be construedas merely illustrative, and not limitations of the preceding disclosurein any way whatsoever. Those skilled in the art will promptly recognizeappropriate variations from the procedures both as to reactants and asto reaction conditions and techniques.

Construction of DNA inserts, plasmids and recombinant viral vectors wascarried out using the standard molecular biology techniques described byJ. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). All therestriction fragments used for the present invention were isolated usingthe “Geneclean” kit (BIO 101 Inc., La Jolla, Calif.).

Example 1 Construction of Cell Line RK13_Pol

Cells and Viruses

Rabbit kidney (RK13) cell was maintained in Earle's minimum essentialmedium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum(FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin (1% Pen/Strep).Equine skin fibroblast cell line (NBL6) was maintained in EMEMsupplemented with 10% FBS, 29 mg/ml L-glutamate, 1% Pen/Strep and 1%nonessential amino acids (Gibco BRL). EHV-1 strains RacL11 and NY03 weregrown on fresh RK13 cells. NY03, harboring a non-neurological form ofDNA polymerase (N752 Pol), was isolated from an aborted foal from a farmin New York in 2003.

Construction of Cell Line RK13 Pol

To support the growth of Pol-negative RacL11 mutant, a rabbit kidneycell line designated RK13_Pol expressing the non-neurological form ofthe polymerase (N752) was first generated. The whole polymerase openreading frame of NY03 was amplified from viral genomic DNA, which wasextracted from virus infected RK13 cells. The polymerase chain reaction(PCR) was performed using Accuprime DNA polymerase (Invitrogen,Carlsbad, Calif., USA) and primer pair PN1/PN2 (Table 1). The amplicon,flanked with a HindIII restriction site at 5′end and a BamHI site aswell as a HA Tag at 3′end, was cloned into plasmid pCDNA3 to generatepCDNA3-Pol. After digestion with PvuI, 4 μg of recombinant plasmidpCDNA3-Pol was linearized and transfected into RK13 cells grown insix-well plates using Lipofectamine 2000 (Invitrogen). Transfected cellswere propagated in EMEM containing 10% FBS and 1.2 mg/ml G418 (Merck). Asingle cell clone in which every cell was shown to express DNApolymerase by indirect immunofluorescence assay (IFA) was selected andtermed RK13_Pol. By IFA using anti-HA MAb, the expression of polymerasein RK13_Pol could be demonstrated (FIG. 3) and shown to be stable in tentimes of passages.

TABLE 1  Primer Sequences Prim- SEQ er ID Name NO: Sequence PN1 165′-CCCAAGCTTgagATGGCGGCGCGCGAACAGG CCA-3′ PN2 175′-CGCGGATCCTTAAGCGTAGTCTGGGACGTCGTA TGGGTAGCTTTGATGGGGAGCTGCTTCT-3′ P118 5′-GCCTGCGTGGAGGAGTATTGGG-3′ P2 19 5′-TAATTGATTACTATTAATAACTATTACACCGGAGGAAGAAAGTCG-3′ P3 20 5′-CAAACTCATCAATGTATCTTAAGGTCTGTGTAAATTTAAAGTGCGA-3′ P4 21 5′-CAAAGGTGCCAGCGTCACATCG-3′ P5 225′-ACGACTTTCTTCCTCCGGTGTAA TAGTTATTAATAGTAATCAATT-3′ P6 235′-CCCTTGCTCACCATGGTGGCGGATCTGACGGTT CACTAAACC-3′ P7 245′-GGTTTAGTGAACCGTCAGATCCGCCACC ATGGTGAGCAAGGG-3′ P8 255′-CGCACTTTAAATTTACACAGAC CTTAAGATACATTGATGAGTTTG-3′ P9 26 5′-TCCCCGCGGATAACTTCGTATAGCATACATTA TACGAAGTTATTAGTTATTAATAGTAATCAAT-3′ P10 275′-CTAGCTAGC ATAACTTCGTATAATGTATGCTA TACGAAGTTATCTTAAGATACATTGATGAGTT-3′ gC-1 28 5′-GACTCTGTCGACGGCCACCGCCGAC-3′ gC-2 295′-CCTGGATCCAGACTCTATTCCCATG-3′ ΔgC-1 30 5′-TTGGCCTATGCGGACGACTT-3′ΔgC-2 31 5′-CCCTTTGGTGCATGGTATGT-3′ Poly1 325′-TCTGG AACTA TCGGC GGTGG C-3′ Poly2 33 5′-CGGGT CTTGA GGAGC ATGTC G-3′

Example 2 Plasmids and Viral Mutagenesis

Generation of Virus Mutants

Conventional homologous recombination strategy was employed for allgenetic manipulations. Two 1.7 kbp flanking fragments on either side ofDNA polymerase were amplified using thermostable Pfu polymerase(Promega) and primer pairs P1/P2 and P3/P4 from RacL11 genome. Anothertwo primer pairs P5/P6 and P7/P8 were used to amplify the HCMV (humancytomegalovirus) promoter and EYFP (enhanced yellow fluorescent protein)gene from plasmid pEYFP-N1, separately. The 5′ends of primers P2 and P5,P3 and P8 as well as P6 and P7 carry homologous sequences of 21-23 bp.With an overlapping PCR using P5 and P8, HCMV promoter and EYFP genewere fused and cloned into plasmid pCR2.1-Topo (Invitrogen) resulting inpCES1. For the construction of shuttle plasmid pCR-Topo-P1P4, the abovetwo homolog arms and EYFP expressing cassette in pCES1 were combined byoverlapping PCR using primers P1/P4 and cloned into pCR2.1-Topo. UsingpCES1 as template and primer pair P9/P10, EYFP cassette flanked withSacII and NheI restriction sites as well as LoxP sequences upstream anddownstream was amplified and cloned into pCR2.1-Topo resulting inplasmid pCES2. To generate plasmid pCR-Topo-gC, a 5.87 kbp gC-containingfragment, which was amplified from RacL11 genome using primers gC-1 andgC-2, was cloned into pCR2.1-Topo. After digestion with SacII and NheI,EYFP cassette was released from pCES2 and transferred to pCR-Topo-gC toreplace gC gene and the recombinant shuttle plasmid was termedTopo-ΔgC-EYFP. Plasmids pCES1, pCES2, pCR-Topo-P1P4 and Topo-ΔgC-EYFPwere identified by digestion and sequencing. The cloning scheme isdepicted in FIG. 2.

For the mutation of DNA polymerase, 1 μg of RacL11 viral DNA and 10 μgof plasmid pCR-Topo-P1P4 were co-transfected into RK13_Pol cells in a6-well plate by calcium phosphate precipitation. Two days posttransfection, green plaques were observed under a fluorescencemicroscope (Axiovert 25, Zeiss). The whole virus was harvested after 2cycles of freeze-thaw and grown on fresh RK13_Pol cells that wereoverlaid with 1.5% methylcellulose in EMEM-2% FBS at 1 hour postinfection. After 3 times of purification, homogenous Pol-negative RacL11mutant, termed L11_ΔPol EYFP, was generated and identified usingrestriction fragment length polymorphism (RFLPs) analyses. To restoreN752 Pol, a 7 kbp fragment, termed p1p4pol, was amplified from EHV-1NY03 genome using Accuprime Tag polymerase and primers P1/P4. Byco-transfection of 1 μg of L11_ΔPol EYFP viral DNA and 4 μg of p1p4polPCR product into RK13 cells, homologous recombination was used again tointroduce N752 Pol variant and RacL11 mutant L11_D752N was obtained. TheD752 to N752 mutation in polymerase was confirmed (FIG. 2D) bysequencing a 780 bp Pol fragment that was amplified from L11_D752N usingsequencing primers poly1 and poly2.

Next steps were to delete gC open reading frame from L11_D752N mutant.Briefly, 1 μg of L11_D752N viral DNA and 10 μg of transfer plasmidTopo-ΔgC-EYFP were co-transfected into RK13 cells. The SacII/NheIfragment (1.2 kbp in length) in gC gene was replaced with EYFP cassetteleading to the generation of recombinant virus L11_D752N ΔgC EYFP. TheEYFP selection marker was excised by expressing Cre recombinase uponco-transfection of L11_D752N ΔgC EYFP and plasmid pCAGGS-NLS/Cre intoRK13 cells. White plaques were purified and a gC-negative,non-neurological RacL11 mutant L11_D752N ΔgC was finally engineered. Bythe homologous recombination between L11_D752N ΔgC EYFP viral DNA andplasmid pCR-Topo-gC, gC gene was repaired, resulting in a gC revertantL11_D752N ΔgC rev (FIG. 2). The deletion of gC gene and the absence ofgC protein were confirmed by PCR identification using primer pairsgC-1/gC-2 and ΔgC-1/ΔgC-2, sequencing, as well as indirectimmunofluorescence assay (IFA).

All the primers used for the construction of plasmids and sequencingwere listed in Table 1.

Indirect Immunofluorescence Assay (IFA)

For the detection of DNA polymerase that was expressed in RK13_Polcells, monoclonal antibody (MAb) directed against HA Tag (H3663, Sigma)was used. RK13_Pol cells grown in a 6-well plate were washed withphosphate-buffered saline (PBS) and fixed in 3.5% paraformaldehyde inPBS for 1 h at room temperature (RT), followed by a 5 min incubation inPBS containing 30 mM glycine and another 5 min permeation in 0.1% TritonX-100 in PBS. After washing with PBS, cells were blocked with PBS-3%bovine serum albumin (BSA) for 30 min at RT. Cells were then incubatedwith the primary antibody at a 1:10000 dilution in PBS-3% BSA for 1 h atRT and extensively washed with PBS. The secondary antibody (AlexaFluor568-conjugated goat anti-mouse IgG, Invitrogen) was added with adilution of 1:2000 in PBS-3% BSA and incubated for 1 h at RT. Afterthorough washing for 3 times of 10 min, fluorescence signal wasinspected under the inverted fluorescence microscope (Axiovert 25,Zeiss).

To confirm the absence of gC protein, RK13 cells were seeded in a 6-wellplate and infected with wild type RacL11, mutant L11_D752N ΔgC or gCrevertant L11_D752N ΔgC rev at a multiplicity of infection (MOI) of0.0001. One hour post infection, viruses were removed and infected cellswere overlaid with 1.5% methylcellulose in EMEM-2% FBS. After 48 h ofincubation at 37° C., cells were fixed and blocked as described above.MAb 1G4 directed against EHV-1 gC protein and B8 against EHV-1 gMprotein were used as the primary antibodies with a dilution of 1:100 and1:200, separately. After incubation with the secondary antibody (AlexaFluor568-conjugated goat anti-mouse IgG) for 1 h, cells were washed andplaques were observed.

Characterization of Virus Mutants

A Pol-negative virus mutant L11_ΔPol EYFP was generated by replacing theauthentic polymerase gene with EYFP via co-transfection of RacL11 viralDNA and shuttle plasmid pCR-Topo-P1P4 into RK13_Pol. L11_ΔPol EYFP wasfound to be able to grow only in RK13_Pol but not in non-complementingRK13 cells (FIG. 4), which confirms that DNA polymerase is essential forvirus growth of EHV-1 in vitro. The mutation from neurological D752 tonon-neurological N752 genotype was then achieved by restoring thenon-neurological polymerase gene of EHV-1 NY03 into L11_ΔPol EYFP. Thesingle amino acid variation was confirmed by sequencing a 780 bpamplicon from the mutant L11_D752N. Based on L11_D752N, the SacII/NheIregion representing a 1222 bp fragment (from nucleotide position 94 to1315) in gC gene was replaced with EYFP, resulting in a gC-negativeintermediate L11_D752N ΔgC EYFP. By the expression of Cre, EYFP cassettewas finally excised, leaving one copy of LoxP sequence (34 bp) betweenthe SacII and NheI restriction sites in the engineered mutant L11_D752NΔgC. To confirm the deletion in gC gene, PCR was performed. While fromthe parental mutant L11_D752N, 5.87 kbp and 1.6 kbp fragments could beamplified using primer pair gC-1/gC-2 and ΔgC-1/ΔgC-2, from L11_D752NΔgC, however, 4.68 kbp and 410 bp fragments were amplified (FIG. 2),suggesting that a deletion of 1.2 kbp in gC gene was present. By IFAusing anti-EHV-1 gC MAb, it could be shown that gC protein wasdetectable in cells infected with either RacL11 or the revertantL11_D752N ΔgC rev, but in cells infected with L11_D752N ΔgC, theexpression of gC protein was abolished (FIG. 5). A gC-negative andnon-neurological RacL11 mutant was thus successfully generated.

Example 3 Characterization of In Vitro Virus Growth

Virus Attachment Assay

Virus attachment assay was carried out as described in Sun et al. (J GenVirol 77, 493-500, 1996) with slight modifications. Monolayers of RK13cells seeded in 6-well plates were cooled for 1 h at 4° C. and infectedwith RacL11, L11_D752N, L11_D752N ΔgC or L11_D752N ΔgC rev with an MOIof 400 plaque-forming units (PFU) per well. The viruses were left toattach for various length of time (0, 15, 30, 60, 120, 240 min) at 4° C.At various time points, infected cells were washed three times with PBSand overlaid with EMEM containing 2% FBS and 1.5% methylcellulose. Afterincubation for 3 days at 37° C., cells were fixed with 10% formaldehydeand stained with 0.3% crystal violet. Plaques were counted and thepercentage of virus attachment at each time point was calculated,relative to time point 240 min that was set to 100% attachment.

Plaque Size and Single-Step Growth Kinetics

To compare the plaque sizes, RK13 cells grown in 6-well plates wereinfected with RacL11 wild type and the mutants L11_D752N, L11_D752N ΔgCor L11_D752N ΔgC rev at an MOI of 0.0001 and overlaid with 1.5%methylcellulose in EMEM containing 2% FBS at 2 hpi. Three dayspost-infection, plaques were visualized by IFA using anti-EHV-1 gM MAbB8. For each virus, 50 plaques were photographed and average plaqueareas were measured by using ImageJ software (available on NIH'sResearch Services Branch website). Plaque sizes induced by RacL11 wereset to 100%. Mean percentage and standard deviation were calculated fromthree independent experiments. For single-step growth kinetics assays,RK13 cells seeded in 24-plates were infected at an MOT of 3. The viruseswere allowed to attach for 1 h at 4° C., followed by a penetration stepof 1.5 h at 37° C. After washing twice with PBS, the infected cells weretreated with ice-cold citrate buffered saline (CBS, pH 3.0) for 3 min toremove virus on the surface. At different time points (0, 4, 8, 12, 24,36, 48 hpi), supernatants and cells were collected separately.Extracellular and cell-associated viral titers were determined usingconventional plaque assays. Single-step growth curves were computed fromthree independent experiments.

In Vitro Growth Properties

The in vitro growth properties of various virus mutants in culturedcells were analyzed. To investigate the impact of gC deletion on bindingability of L11_D752N ΔgC to target cells, attachment assay wasperformed. As expected, L11_D752N ΔgC was shown to bind less efficientlyto RK13 cells when compared with either wild type RacL11, parental virusL11_D752N or the revertant L11_D752N ΔgC rev (FIG. 6). The ability ofL11_D752N ΔgC to spread from cell to cell was determined by comparingplaque sizes. Whereas no significant difference could be observedbetween wild type, parental virus and gC revertant, the relative plaquearea formed by L11_D752N ΔgC, however, was 10% larger (p<0.0001) thanthose of others (FIG. 7), indicating that with the absence of gC, thecell-to-cell spread of EHV-1 is even more efficient. With respect togrowth kinetics, the in vitro replication of L11_D752N ΔgC was lesseffective as demonstrated by extracellular and intracellular titers thatwere reduced by approximately 20-fold and 10-folds, respectively,relative to wild type, parental virus or gC revertant (FIG. 8). Thevirus titers in cell culture supernatants infected with wild typeRacL11, parental virus L11_D752N or the repaired L11_D752N ΔgC revreached the value of intracellular titers at approximately 12 hourspost-infection. In L11_D752N ΔgC infected cells, however, a delayedrelease of infectious progeny was observed by that the extracellulartiter crossed intracellular titer at 16-18 hours post-infection. Fromthese results, it could be concluded that the in vitro growth ofL11_D752N ΔgC was impaired in virus binding and egress though it couldform slightly larger plaques.

Example 4 Vaccination of Mice

Wild type RacL11 as well as the mutants L11_D752N, L11_D752N ΔgC andL11_D752N ΔgC rev were purified by ultracentrifuge for 1 h at 27,000rpm. The pellets were suspended in PBS, titrated on fresh RK13 cells,aliquoted and stored at −70° C. until use. Three-week-old female BALB/cmice (Harlan) were randomly allocated into five groups of 16 mice eachand left to acclimate to each other for one week in the BiosafetyLevel-2 facility. On day 0, all mice were weighted, anesthetized with0.1 mL/10 g Xylazine/Ketamine and four groups were inoculatedintranasally (IN) with each virus at a dose of 1×10⁵ PFU in 20 μL ofPBS. The last group was used as a negative control and received 20 μL ofPBS. Bodyweights of each mouse was inspected until day 14 postinoculation (p.i.). Three mice from each group were euthanized on day 2and day 4. The lung was removed, part of which was homogenized and usedfor determination of virus titers by standard titration on RK13 cells,and the rest was fixed with 10% formaldehyde and processed forhistopathological analysis. On day 28, mice were challenged with wildtype RacL11 at a dose of 1×10⁵ PFU by the intranasal route. Data ofindividual bodyweights were collected for two weeks. On day 2 and day 4post challenge (p.c.), three mice of each group were euthanized toremove the lung. Virus titers in the lung tissues were determined onfresh RK13 cells after homogenization.

On day 2 p.i., mean virus titers in lungs infected with RacL11,L11_D752N or L11_D752N ΔgC rev reached 5366 PFU/mg, 3450 PFU/mg and 4800PFU/mg, respectively (FIG. 9), between which no statisticallysignificant difference was observed (p≧0.442). This result indicatedthat the single amino acid variation from D752 to N752 in DNA polymerasedoes not impair the in vivo growth of EHV-1 in mice, which wasconsistent with previous findings (Goodman et al., 2007). In contrast,the in vivo replication of the gC-negative mutant L11_D752N ΔgC wassignificantly less effective (p<0.001), with a mean virus titer of only0.45 PFU/mg in lung tissues. On day 4 p. i., no virus could be recoveredfrom L11_D752N ΔgC infected mice. With respect to histopathologicalchanges on day 2 p. i., the lungs of mice infected with either wild typeRacL11 or the polymerase mutant L11_D752N showed mild suppurativepneumonia accompanied by neutrophilic infiltration, increasedperivascular edema and increased number of inflammatory cells(predominantly lymphocytes) in lymph vessels and perivascular area,whereas no abnormality was detected in lungs infected with L11_D752N ΔgCand PBS (FIG. 10). As a result of the inflammatory response, continuousbody weight loss of mice infected with RacL11, L11_D752N or the rescuantvirus L11_D752N ΔgC rev was observed till day 3 p. i. The body weight ofmice infected with L11_D752N ΔgC, however, did not exhibit apparentreduction. On day 28 p. i., all the mice were challenged intranasallywith RacL11. Two days post challenge (p. c.), RacL11 replicated to atiter of 2100 PFU/mg in lungs of mock-inoculated mice, but only 18.8PFU/mg in L11_D752N ΔgC group (FIG. 9). The virus titers in lungs ofmice inoculated with RacL11, L11_D752N or L11_D752N ΔgC rev were evenlower (0.5, 0.9 and 1.3 PFU/mg, FIG. 9) on day 2 p. c., which was,however, at the expense of high growth efficiency and pathogenicity invivo. On day 4 p. c., resolution of virus from L11_D752N ΔgC group wasnot successful. On the basis of these results we concluded that thegC-negative, nonneurological mutant L11_D752N ΔgC is severely attenuatedand apathogenic for mice, but can confer protective immunity.

Example 5 Vaccination of Horses

Horses (6 to 8 month old foals) were grouped into three groups A, B andC. Group A was treated with live attenuated EHV-1 virus having gC genedeleted (L11_D752N ΔgC). Group B was treated with live attenuated EHV-1virus (RacL11). Group C was control group. Vaccination and samplingswere done according to the schedule shown in Table 2.

TABLE 2 Samples for Post- Vaccination vaccine challenge Blood NasalGroup 6.3 log₁₀DICC₅₀ shedding Challenge examination sampling swabbing AIntramuscular D −1, D 3, D 4, D 42 Daily from D −1, D 7, D −1, n = 8route D 5, D 6, D 7, EHV1 D 42 to D 13, D 27, D 41, D 0: 2.4 ml D 10 andD 13. strain D 56 D 41, D 43, daily D 28: 2.0 ml D 27, D 31, 10^(5.0) D44, D 45, from B D 0 and D 28 D 32, D 33, TCID50/ D 46, D 47, D 43 to n= 8 Intramuscular D 34, D 35, nostril D 48, D 49, D 49, route D 38 and D41 D 52 and D 52 and Dose = 1.5 ml D 56 D 56 C Not vaccinated D −1 and D27 n = 8

Both vaccines were well tolerated by the foals. The vaccine virusstrains could not be detected in any sample by virus isolation. Controlhorses did not seroconvert up to the time of challenge as evidenced bythe absence of SN and CF antibody titers to EHV-1. Horses from group Aand B mounted a SN response to first vaccination which was boosted aftersecond vaccination. CF antibody titers in both groups of horses remainedlow/undetectable after first vaccination and increased after secondvaccination, but never reached high levels (FIG. 11).

Clinical signs after challenge in the control horses included atri-phasic fever response and respiratory disease characterized bymoderate to severe nasal discharge. In contrast, hyperthermia was onlysporadically found in both vaccinated groups of horses and nasaldischarge was reduced in severity and duration. A notable effect ofvaccination was the complete absence of viremia after challenge in 5/8horses from both group 1 and 2 (FIG. 12). Also the duration of viremiawas reduced in the vaccinated compared to the control horses. FIG. 13shows that group A had significantly reduced virus shedding in the nasalswabs, whereas nasal shedding in group B horses was more variable, butreduced compared to the control horses.

Example 6 Sequencing of RacL11 gC Gene

The gC gene of RacL11 was sequence and is represented by SEQ ID NO:34and SEQ ID NO:35 for DNA and protein sequence, respectively.

It will be apparent that the precise details of the methods describedmay be varied or modified without departing from the spirit of thedescribed disclosure. We claim all such modifications and variationsthat fall within the scope and spirit of the claims below.

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention.

What we claim is:
 1. A composition comprising a recombinant EquineHerpesvirus-1 (EHV-1), wherein the EHV-1 comprises a mutated gene whichencodes a non-functional Glycoprotein C (gC) wherein the gC gene isdeleted, and wherein the EHV-1 further comprises a DNA polymerase (Pol)comprising an asparagine (N) at the amino acid position 752 from thestarting methionine.
 2. The composition of claim 1, further comprising apharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant,or excipient.
 3. A recombinant EHV-1 comprising a mutated gC geneencoding a protein wherein the gC gene is deleted, and a DNA polymerase(Pol) gene encoding a Pol comprising an asparagine (N) at the amino acidposition 752 from the starting methionine.
 4. A method of vaccinating ananimal comprising at least one administration of the composition orrecombinant EHV-1 of claim 1 or
 3. 5. The method of claim 1, wherein themethod comprises a prime-boost administration regime.
 6. A method ofeliciting a protective response in an animal against Herpesviruscomprising administering to the animal the composition or recombinantEHV-1 of claim 1 or 3 and a pharmaceutically or veterinarily acceptablecarrier, adjuvant, excipient or vehicle.
 7. The method of claim 4 or 6,wherein the animal is equine.
 8. The composition of claim 1, wherein theEHV-1 is derived from an EHV-1 strain selected from the group consistingof the RacH strain, the RacL strain, the Ab4 strain, the V592 strain,the Kentucky D strain, the 438/77 strain, the AB69 strain, the EHV-1NY03 strain, and a combination thereof.
 9. The recombinant EHV-1 ofclaim 3, wherein the EHV-1 is derived from an EHV-1 strain selected fromthe group consisting of the RacH strain, the RacL strain, the Ab4strain, the V592 strain, the Kentucky D strain, the 438/77 strain, theAB69 strain, the EHV-1 NY03 strain, and a combination thereof.